PROJECT SUMMARY This proposal seeks to elucidate how G protein-coupled receptors (GPCRs) are regulated by naturally produced ligands and addictive drugs, focusing at the cell biological level as the critical bridge linking molecular and systems-level events and understanding. GPCRs represent the largest family of signaling receptors, comprise in aggregate the largest class of therapeutic drug targets, and mediate directly or indirectly the effects of all addictive drugs. Accordingly, while addictive drug action in the CNS is our particular focus, the proposed studies have broad potential application across GPCR family members and pathophysiological processes. Our over-arching goal is to develop a fundamental understanding of GPCR regulation, discovering the underlying cell biology and then elucidating its molecular basis, and through this path discover new targets and strategies for potential therapeutic manipulation of addictive and other complex brain disorders that are characterized by underlying disturbances of GPCR signaling or GPCR-dependent physiological regulation. Progress in the previous funding period focused on defining sites of regulatory phosphorylation in the mu opioid receptor, accomplishing this in intact human cells expressing the full spectrum of endogenous kinases at native levels. We defined two critical regions of phosphorylation and carried out detailed cell biological analysis of one of them, both in a heterologous cell model and a physiologically relevant population of CNS- derived neurons. During the course of these studies we made some major unanticipated progress, including development of conformational biosensor technology, discovery of GPCR signaling via heterotrimeric G proteins from endosomes, and discovery of an unprecedented behavior of arrestin proteins suggesting a new cellular operating mode of arrestins, downstream of and after dissociating from an activating GPCR. The proposed studies seek to develop and extend these fundamental new observations and develop them to the point of rational consideration as new molecular targets and cell-based strategies for therapeutics.